5/11/2021 USP-NF Hesperidin

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© 2021 USPC

Hesperidin

C28H34O15 610.57
4H-1-Benzopyran-4-one, 7-[[6-O-(6-deoxy-α-L-mannopyranosyl)-β-D-glucopyranosyl]oxy]-2,3-dihydro-5-hydroxy-2-(3-hydroxy-4-
methoxyphenyl)-, (2S)-;
(2S)-5-Hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4-oxo-3,4-dihydro-2H-chromen-7-yl 6-O-(α-L-rhamnopyranosyl)-β-D-
glucopyranoside [ 520 - 26 -3].

DEFINITION
Hesperidin contains NLT 90.0% and NMT 102.0% of hesperidin (C28H34O15), calculated on the dried basis.

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IDENTIFICATION
Change to read:
• A. ▲SPECTROSCOPIC IDENTIFICATION TESTS 〈197〉, Infrared Spectroscopy: 197K▲ (CN 1-MAY-2020)
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• B. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay.

ASSAY
• PROCEDURE
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Solution A: Dilute 5 mL of glacial acetic acid with water to 1000 mL.

Mobile phase: Methanol and Solution A (30:70)
System suitability solution: 0.2 mg/mL each of USP Isonaringin RS, USP Hesperidin RS, USP Neohesperidin RS, USP Diosmin RS, and
USP Didymin RS in dimethyl sulfoxide
Standard solution: 1.0 mg/mL of USP Hesperidin RS in dimethyl sulfoxide
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Sample solution: 1.0 mg/mL of Hesperidin in dimethyl sulfoxide

Chromatographic system
(See Chromatography 〈621〉, System Suitability.)
Mode: LC
Detector: UV 284 nm
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Column: 3.9-mm × 15-cm; 4-µm packing L1

Column temperature: 40°
Flow rate: 1.0 mL/min
Injection volume: 10 µL
Run time: At least 5 times the retention time of hesperidin
System suitability
Samples: System suitability solution and Standard solution
[NOTE—The relative retention times for hesperidin and its related compounds are shown in Table 1.]
Suitability requirements
Resolution: NLT 1.8 between hesperidin and neohesperidin, System suitability solution
Relative standard deviation: NMT 2.0%, Standard solution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of hesperidin (C28H34O15) in the portion of Hesperidin taken:

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5/11/2021 USP-NF Hesperidin

Result = (rU/rS) × (CS/CU) × 100

rU = peak response from the Sample solution

rS = peak response from the Standard solution

CS = concentration of USP Hesperidin RS in the Standard solution (mg/mL)

CU = concentration of Hesperidin in the Sample solution (mg/mL)

Acceptance criteria: 90.0%–102.0% on the dried basis

IMPURITIES
• RESIDUE ON IGNITION 〈281〉
Sample: 1.0 g
Acceptance criteria: NMT 0.2%
• RELATED COMPOUNDS
Solution A, Mobile phase, System suitability solution, Chromatographic system, and System suitability: Proceed as directed in the
Assay.
Standard solution: 0.05 mg/mL of USP Hesperidin RS in dimethyl sulfoxide
Sample solution: 1.0 mg/mL of Hesperidin in dimethyl sulfoxide
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of each impurity in the portion of Hesperidin taken:

Result = (rU/rS) × (CS/CU) × F × 100

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rU = peak response of each impurity from the Sample solution

rS = peak response of hesperidin from the Standard solution

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CS = concentration of USP Hesperidin RS in the Standard solution (mg/mL)

CU = concentration of Hesperidin in the Sample solution (mg/mL)

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F = correction factor for each individual impurity (see Table 1)

Acceptance criteria: See Table 1. [NOTE—Disregard any impurity less than 0.1%.]

Table 1
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Acceptance Criteria, NMT

Name Relative Retention Time Correction Factor (F) (%)

Eriocitrina 0.4 1.00 1.0

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Isonaringin 0.7 1.07 4.0

Hesperidin 1.0 — —

Neohesperidin 1.2 0.91 1.0

Diosmin 1.5 1.67 1.0

Naringeninb 2.6 0.51 1.0

Didymin 3.0 1.02 3.0

Hesperetinc 3.8 0.45 1.0

Any unspeci ed impurity — 1.00 1.0

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5/11/2021 USP-NF Hesperidin

Acceptance Criteria, NMT

Name Relative Retention Time Correction Factor (F) (%)

Total unspeci ed impurities — — 3.0

Total impurities — — 10.0

a
  (S)-5-Hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4-oxo-3,4-dihydro-2H-chromen-7-yl 6-O-(α-L-rhamnopyranosyl)-β-D-glucopyranoside.
b  (2S)-5,7-Dihydroxy-2-(4-hydroxyphenyl)chroman-4-one.
c  (S)-5,7-Dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chroman-4-one.

SPECIFIC TESTS
• LOSS ON DRYING 〈731〉
Analysis: Dry at 105° for 4 h.
Acceptance criteria: NMT 5.0%
• MICROBIAL ENUMERATION TESTS 〈2021〉: The total bacterial count does not exceed 103 cfu/g, and the total combined molds and yeasts
count does not exceed 102 cfu/g.
• ABSENCE OF SPECIFIED MICROORGANISMS 〈2022〉, Test Procedures, Test for Absence of Salmonella SpeciesandTest for Absence of Escherichia
coli: Meets the requirements

ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in well-closed, tight containers.
• USP REFERENCE STANDARDS 〈11〉
USP Didymin RS

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(RS)-5-Hydroxy-2-(4-methoxyphenyl)-4-oxo-3,4-dihydro-2H-chromen-7-yl 6-O-(α-L-rhamnopyranosyl)-β-D-glucopyranoside.
C28H34O14 594.57
USP Diosmin RS
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USP Hesperidin RS
USP Isonaringin RS
(RS)-5-Hydroxy-2-(4-hydroxyphenyl)-4-oxo-3,4-dihydro-2H-chromen-7-yl 6-O-(α-L-rhamnopyranosyl)-β-D-glucopyranoside.
C27H32O14 580.54
USP Neohesperidin RS (S)-5-Hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4-oxo-3,4-dihydro-2H-chromen-7-yl 2-O-(α-L-rhamnopyranosyl)-β-D-
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glucopyranoside. C28H34O15 610.57

Auxiliary Information- Please check for your question in the FAQs before contacting USP.
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Topic/Question Contact Expert Committee

HESPERIDIN Maria Monagas NBDS2020 Non-botanical Dietary

Scienti c Liaison Supplements
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Chromatographic Database Information: Chromatographic Database

Most Recently Appeared In:

Pharmacopeial Forum: Volume No. 41(2)

Page Information:

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USP41-NF36 - 4703

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